US7300784B2 - Nitrile hydratase and a method for producing amides - Google Patents

Nitrile hydratase and a method for producing amides Download PDF

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US7300784B2
US7300784B2 US10/469,427 US46942704A US7300784B2 US 7300784 B2 US7300784 B2 US 7300784B2 US 46942704 A US46942704 A US 46942704A US 7300784 B2 US7300784 B2 US 7300784B2
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nitrile hydratase
nitrile
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Toru Nagasawa
Akinobu Matsuyama
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Daicel Corp
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/02—Amides, e.g. chloramphenicol or polyamides; Imides or polyimides; Urethanes, i.e. compounds comprising N-C=O structural element or polyurethanes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/88—Lyases (4.)

Definitions

  • the present invention relates to a novel nitrile hydratase and a method for producing amide compounds from nitrile compounds using the nitrile hydratase.
  • nitrile hydratases are derived form the following microorganisms. All of the nitrile hydratases are enzymes consisting of two types of heterogeneous subunits.
  • Rhodococcus (Examined Published Japanese Patent Application (JP-B) NO. Hei 3-54558)
  • Rhodococcus (Unexamined Published Japanese Patent Application (JP-A) NO. Hei 2-119778)
  • the expression level of nitrile hydratase activity is not high enough with E. coli transformed with an expression plasmid containing an insert fragment of any nitrile hydratase gene described in these patent publications; the nitrile-hydrating activity per weight of cells of the transformant is lower than that of the original microorganism from which the gene is derived (Ikehata, O., Nishiyama, M., Horinouchi, S. and Beppu, T. “Primary structure of nitrile hydratase deduced from the nucleotide sequence of a Rhodococcus speices N-774 and its expression in Escherichia coli ” Eur. J. Biochem.
  • nitrile hydratase which exhibits very high nitrile hydration activity, has been found in Achromobacter xerosis (IFO 12668), and the gene encoding the enzyme has been cloned. Further, the gene was introduced into E. coli using an expression plasmid, and nitrile hydratase was overproduced with the resulting transformant (JP-A Hei 8-266277).
  • This report shows only examples of acrylamide and ⁇ -hydroxyisobutylamide production, and thus there is no description on the activity of this enzyme toward 2-hydroxy-4-methylthiobutyronitrile in the report.
  • HMBN 2-hydroxy-4-methylthiobutyronitrile
  • HMBAm 2-hydroxy-4-methylthiobutylamides
  • the enzymes having the nitrile hydratase activity capable of converting nitrile compounds to amide compounds have a problem that the enzymes readily lose their own enzymatic activities due to the presence of the nitrile compound as starting material or amide compound as the product. If the concentration of nitrile compound is raised in order to increase the rate of amidation, the nitrile hydratase is readily inactivated in a short period of time, and thus it is hard to obtain amide compound as the reaction product in a desired period of time. In addition, the amide compounds as the products also readily inactivate the nitrile hydratase, and thus it is difficult to obtain a high concentration of amide compound.
  • aldehydes are linked to proteins and can inactivate the enzymatic activity (see Chemical Modification of Proteins, G. E. Means et al., Holden-Day, 125(1971)).
  • hydrocyanic acid cyanide
  • aldehyde and cyanide produced from ⁇ -hydroxynitrile as the starting material can be the cause of decreased enzymatic activity. Because of a problem that the enzyme is inactivated in a short period of time in the enzymatic hydration or hydrolysis of ⁇ -hydroxy nitrile, it was difficult to obtain a high concentration of ⁇ -hydroxyamide with high productivity.
  • An objective of the present invention is to provide a nitrile hydratase having high nitrile-hydrating activity. Another objective of the present invention is to provide a stable nitrile hydratase capable of maintaining the high enzymatic activity over a long period of time. In addition, still another important objective of the present invention is to provide a nitrile hydratase capable of also using 2-hydroxy-4-methylthiobutyronitrile as a substrate.
  • Another objective of the present invention is to provide the gene encoding nitrile hydratase having high nitrile hydration activity, recombinant plasmid containing the gene and transformant containing the recombinant plasmid.
  • yet another objective of the present invention is to provide a method for producing the corresponding amides from nitrile using the transformant expressing high nitrile hydration activity.
  • the present inventors strenuously studied to achieve the above-mentioned objectives, and found nitrile hydratase having very high nitrile hydration activity in microorganisms belonging to the genus Rhodococcus ( Rhodococcus sp.). Then, the present inventors confirmed that the nitrile hydratase was a novel enzyme usable for achieving the above-mentioned objectives, and thus completed the present invention.
  • the inventors then cloned the gene encoding the enzyme by recombinant DNA technology, and prepared E. coli transformed with the expression plasmid comprising an expression vector containing the isolated gene as an insert. Further, the present inventors succeeded in the large-scale production of the nitrile hydratase with the obtained transformant and thus completed the present invention.
  • the present invention provides the following nitrile hydratase, polynucleotide encoding the enzyme, method for producing the enzyme and method for producing amides using the enzyme of the present invention.
  • the nitrile hydratase of the present invention has the following physicochemical properties (a) and (b):
  • the activity of hydrating the nitrile group of nitrile compound and converting it to an amide group is referred to as “nitrile hydratase activity”.
  • an enzyme capable of acting on the compound of the following formula (1) and producing the amide compound of formula (3) is designated as “nitrile hydratase”.
  • R represents substituted or unsubstituted alkyl group, substituted or unsubstituted alkenyl group, substituted or unsubstituted cycloalkyl group, substituted or unsubstituted alkoxy group, substituted or unsubstituted aryloxy group, substituted or unsubstituted saturated or unsaturated heterocyclic group).
  • the nitrile hydratase of the present invention can act on 2-hydroxy-4-methylthiobutyronitrile and produce 2-hydroxy-4-methylthiobutyroamide.
  • the nitrile hydratase activity of the present invention can be confirmed as follows. At first, an enzyme sample is added to a 0.1 M potassium phosphate buffer (pH 6.5) containing 10% v/v 2-hydroxy-4-methylthiobutyronitrile (HMBN) as the substrate. Instead of the enzyme sample, microorganism cells and crude enzyme are also usable. After addition of enzyme, the solution is incubated at 20° C. for 15 minutes. The reaction solution is then combined with an excess volume of 0.1% (v/v) phosphate solution, and the mixture is shaken vigorously to stop the reaction. The reaction product can be analyzed by HPLC.
  • HMBN 2-hydroxy-4-methylthiobutyronitrile
  • 1 U of nitrile hydratase was defined as the amount of enzyme capable of producing 1 ⁇ mol nicotinamide at 20° C. for 1 minute in a reaction solution with a standard composition; 1 U was defined as the amount of enzyme capable of producing 1 ⁇ mol HMBAm at 20° C. for 1 minute in a reaction solution with a standard composition.
  • the enzymatic activity can be assayed by the procedure as described in Examples. Further, protein quantification is carried out by the dye-binding method using a protein assay kit from Bio-Rad.
  • cyanide resistance means that the enzyme retains 40% or more nitrile hydratase activity when treated at 20° C. for 30 minutes in the presence of 1 mM cyanide ion.
  • the enzyme retains 10% or more nitrile hydratase activity after treated at 20° C. for 30 minutes in the presence of 5 mM cyanide ion, one can state that the enzyme is cyanide-resistant.
  • nitrile hydratase having the following physicochemical properties (1)-(7):
  • the molecular weight is approximately 110,000 Da when determined by gel filtration
  • the enzyme is separated to two subunits of 26.8 kDa and 29.5 kDa by SDS-polyacrylamide gel electrophoresis.
  • the enzyme acts on the nitrile group of nitrile compound.
  • the activity of hydrating nitrile group is maximized at pH 5.5-6.5.
  • the activity of hydrating nitrile group is maximized at 40-45° C.
  • the enzyme is stable within pH 4-9.
  • the enzyme retains 70% or more activity after heat-treated at 50° C. for 30 minutes.
  • the enzyme is inhibited by HgCl 2 , AgNO 3 , hydroxylamine or phenylhydrazine.
  • the present invention provides nitrile hydratase having the following physicochemical properties (c) and/or (d) in addition to the above-mentioned physicochemical properties (a) and (b), or (1)-(7)
  • the enzyme uses 2-hydroxy-4-methylthiobutyronitrile as the substrate and produces 2-hydroxy-4-methylthiobutyroamide.
  • the enzyme is stabilized by divalent metal ions.
  • the substrate specificity of nitrile hydratase can be determined by the method for testing the activity nitrile hydratase as described above.
  • “stabilized by divalent metal ions” means that the enzymatic activity is not substantially reduced even when the enzyme is incubated with 1.8 w/w % 2-hydroxy-4-methylthbutyronitrile in the presence of a divalent metal ion for 20 minutes.
  • the nitrile hydratase of the present invention can maintain 110% or higher activity under these conditions.
  • the above-mentioned divalent metal ion includes nickel ion and cobalt ion. These divalent metal ions enhance the enzymatic activity of nitrile hydratase of the present invention at a concentration of 0.1 mM-1 M, generally 0.5-100 mM, preferably 1-10 mM.
  • the nitrile hydratase of the present invention can be purified from microorganisms capable of producing the enzyme by commonly used protein purification methods.
  • the above microorganisms can be cultured in a standard bacterial culture medium.
  • Some compounds for inducing the expression of nitrile hydratase can be added to the medium.
  • the addition of a nitrile compound or amide compound can enhance the activity of nitrile hydratase. More specifically, acetonitrile, acetamide and such can be used as the enzyme-inducer.
  • Microorganisms capable of producing the enzyme are sufficiently grown, and then the cells are harvested.
  • the cells are lysed in an appropriate buffer to prepare cell-free extract.
  • the buffer can contain a reducing agent such as 2-mercaptoethanol, protease inhibitor such as phenylmethanesulfonyl fluoride (PMFS).
  • the nitrile hydratase can be purified from the cell-free extract by fractionation based on the protein solubility and appropriate combinations of various chromatographic procedures.
  • fractionation method for example, precipitation with an organic solvent such as acetone and dimethylsulfoxide or salting out with ammonium sulfate can be used.
  • organic solvent such as acetone and dimethylsulfoxide
  • salting out with ammonium sulfate can be used.
  • known chromatographic methods include cation-exchange chromatography, anion-exchange chromatography, gel filtration, hydrophobic chromatography as well as many procedures of affinity chromatography using dye, antibody and others.
  • the nitrile hydratase of the present invention can be purified as an electrophoretically homogeneous polypeptide, for example, by hydrophobic chromatography using phenyl-TOYOPEARL, anion-exchange chromatography using DEAE-Sepharose, hydrophobic chromatography using butyl-TOYOPEARL, affinity chromatography using Blue-Sepharose, gel filtration using Superdex 200, and others.
  • Rhodococcus sp. Cr4 is a suitable microorganism for producing the nitrile hydratase of the present invention.
  • Rhodococcus sp. Cr4 has been deposited under the accession number of FERM BP-6596 in the International Patent Organism Depositary.
  • Rhodococcus sp. Cr4 International Deposition of Rhodococcus sp. Cr4:
  • the nitrile hydratase of the present invention is that can be obtained from Rhodococcus sp.
  • Cr4 is a novel enzyme having the above-mentioned physicochemical properties (a)-(d) and (1)-(7).
  • the structural feature is described above as the physicochemical property (1); the enzyme is a hetero-dimeric polypeptide consisting of ⁇ -subunit of 26.8 kDa and ⁇ -subunit of 29.5 kDa, determined by SDS-PAGE.
  • the amino acid sequence of ⁇ -subunit is shown in SEQ ID NO: 2 (226 amino acid residues) and the amino acid sequence of ⁇ -subunit, in SEQ ID NO: 4 (207 amino acid residues).
  • the present invention provides the following substantially pure protein complex having the nitrile hydratase activity.
  • the present invention relates to a substantially pure protein complex between polypeptide comprising the amino acid sequence of SEQ ID NO: 2 and polypeptide comprising the amino acid sequence of SEQ ID NO: 4. Further the present invention includes a homologue of the protein complex between polypeptide comprising the amino acid sequence of SEQ ID NO: 2 and polypeptide comprising the amino acid sequence of SEQ ID NO: 4.
  • substantially pure as used herein in reference to a given protein, polypeptide, or protein complex means that the protein, polypeptide, or protein complex is substantially free from other biological macromolecules.
  • the substantially pure protein, polypeptide, or protein complex is at least 75% (e.g., at least 0.80, 85, 95, or 99%) pure by dry weight. Purity can be measured by any appropriate standard method known in the art, for example, by column chromatography, polyacrylamide gel electrophoresis, or HPLC analysis.
  • the protein complex of the present invention is a nitrile hydratase that can be expressed by genetic recombination techniques using isolated polynucleotides encoding the nitrile hydratase of the present invention.
  • the polynucleotides encoding the nitrile hydratase of the present invention can be isolated, for example, by the following method.
  • the nitrile hydratase provided by the present invention consists of ⁇ -subunit encoded by the polynucleotide shown in any one of (A)-(E), and ⁇ -subunit encoded by the polynucleotide shown in any one of (a)-(e), and is a protein complex having the following physicochemical properties (i) and (ii).
  • the enzyme uses 2-hydroxy-4-methylthiobutyronitrile as the substrate and produces 2-hydroxy-4-methylthiobutyroamide.
  • the polynucleotide shown in any one of the following (A)-(E) can be used as the gene encoding ⁇ -subunit constituting the protein complex of the present invention.
  • the polynucleotide shown in any one of (A)-(E) is useful for expressing the ⁇ -subunit of nitrile hydratase of the present invention:
  • polynucleotide shown in any one of the following (a)-(e) can be used as the gene encoding ⁇ -subunit constituting the protein complex of the present invention.
  • the polynucleotide shown in any one of (a)-(e) is useful for expressing the ⁇ -subunit of nitrile hydratase of the present invention:
  • polypeptide comprising the amino acid sequence of SEQ ID NO: 4, which contains one or more amino acid substitutions, deletions, insertions and/or additions;
  • the present invention relates to isolated polynucleotides encoding subunits of the nitrile hydratase and homologues thereof.
  • an “isolated polynucleotide” is a polynucleotide the structure of which is not identical to that of any naturally occurring polynucleotide or to that of any fragment of a naturally occurring genomic polynucleotide spanning more than three separate genes.
  • the term therefore includes, for example, (a) a DNA which has the sequence of part of a naturally occurring genomic DNA molecule in the genome of the organism in which it naturally occurs; (b) a polynucleotide incorporated into a vector or into the genomic DNA of a prokaryote or eukaryote in a manner such that the resulting molecule is not identical to any naturally occurring vector or genomic DNA; (c) a separate molecule such as a cDNA, a genomic fragment, a fragment produced by polymerase chain reaction (PCR), or a restriction fragment; and (d) a recombinant nucleotide sequence that is part of a hybrid gene, i.e., a gene encoding a fusion polypeptide.
  • the invention provides an isolated polynucleotide that encodes a polypeptide described herein or a fragment thereof.
  • the isolated polynucleotide includes a nucleotide sequence that is at least 60% identical to the nucleotide sequence shown in SEQ ID NO: 1 or 3. More preferably, the isolated nucleic acid molecule is at least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 0.93%, 94%, 0.95%, 96%, 97%, 98%, 99%, or more, identical to the nucleotide sequence shown in SEQ ID NO: 1 or 3.
  • an isolated polynucleotide which is longer than or equivalent in length to the reference sequence e.g., SEQ ID NO: 1 or 3
  • the comparison is made with the full length of the reference sequence.
  • the isolated polynucleotide is shorter than the reference sequence, e.g., shorter than SEQ ID NO: 1 or 3
  • the comparison is made to segment of the reference sequence of the same length (excluding any loop required by the homology calculation).
  • each of the nucleotide sequences of polynucleotides encoding ⁇ -subunit and ⁇ -subunit of nitrile hydratase of the present invention as well as the amino acid sequences encoded by the nucleotide sequences are novel.
  • the genes of interest can be obtained form the deposited microorganism as described above.
  • the genes can be obtained by PCR or screening with hybridization technique.
  • the full-length genes can also be obtained by chemical DNA synthesis.
  • the nitrile hydratase gene derived from other organisms can be isolated by performing, under stringent conditions, hybridization to polynucleotides prepared from other organisms, using the above nucleotide sequence or a partial sequence thereof as a probe.
  • polynucleotide capable of hybridizing under stringent conditions means a polynucleotide capable of hybridizing to a polynucleotide, which has a nucleotide sequence selected from the nucleotide sequences of SEQ ID NO: 1 ( ⁇ -subunit) and SEQ ID NO: 3 ( ⁇ -subunit), as a probe, for example, with an ECL direct nucleic acid labeling and detection system (Amersham Pharmacia Biotech) under the conditions as described in the manual (wash: with a primary wash buffer containing 0.5 ⁇ SSC at 42° C.).
  • a polynucleotide that hybridizes under high stringency conditions to the nucleotide sequence of SEQ ID NO: 1 or a segment thereof as described herein.
  • “High stringency conditions” refers to hybridization in 6 ⁇ SSC at about 45° C., followed by one or more washes in 0.2 ⁇ SSC, 0.1% SDS at 65° C.
  • the nucleotide sequence constituting the probe polynucleotide can be prepared as one or more sequences consisting of arbitrary but at least consecutive 20 nucleotides, preferably at least 30 consecutive nucleotides, for example, consecutive 40, 60 or 100 nucleotides selected from the above-mentioned nucleotide sequences.
  • PCR primers can be designed from regions exhibiting high homology.
  • the gene encoding nitrile hydratases can be isolated from various organisms by PCR using such primers and chromosomal DNA or cDNA as a template.
  • the enzyme in the method of the present invention, it is possible to use not only the natural enzyme but also an enzyme comprising the amino acid sequence of the natural enzyme in which one or more amino acids have been substituted, deleted, and/or inserted, when the enzyme can form the protein complex having the above-mentioned physicochemical properties (a) and (b) or (1) to (7).
  • One skilled in the art can modify the structure of the polypeptide, for example, via introducing mutations of appropriate substitutions, deletions, insertions and/or additions by site-specific mutagenesis (Nucleic Acid Res. 10, pp. 6487 (1982); Methods in Enzymol. 100, pp.
  • amino acid mutations can be generated in nature.
  • the enzyme having artificial amino acid mutations but also the enzyme containing spontaneous amino acid mutations can be used in the method of the present invention.
  • the number of amino acids that are mutated is not particularly restricted, as long as the enzyme can form the protein complex having the above-mentioned physicochemical properties (a) and (b) or (1) to (7). Normally, it is within 50 amino acids, preferably within 30 amino acids, more preferably within 10 amino acids, and even more preferably within 3 amino acids.
  • the site of mutation may be any site, as long as the enzyme can form the protein complex having the above-mentioned physicochemical properties (a) and (b) or (1) to (7).
  • amino acid substitution is preferably mutated into different amino acid(s) in which the properties of the amino acid side-chain are conserved.
  • a “conservative amino acid substitution” is a replacement of one amino acid residue belonging to one of the following groups having a chemically similar side chain with another amino acid in the same group. Groups of amino acid residues having similar side chains have been defined in the art.
  • amino acids with basic side chains e.g., lysine, arginine, histidine
  • acidic side chains e.g., aspartic acid, glutamic acid
  • uncharged polar side chains e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine
  • nonpolar side chains e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan
  • beta-branched side chains e.g., threonine, valine, isoleucine
  • aromatic side chains e.g., tyrosine, phenylalanine, tryptophan, histidine
  • the gene encoding a polypeptide having homology to the amino acid sequence of each subunit of nitrile hydratase can also be used for the present invention, when the product can form the protein complex having the above-mentioned physicochemical properties (a) and (b) or (1)-(7).
  • the genes can be obtained by using protein homology search. Such homology search can be carried out, for example, by using the following publicly known databases:
  • DNA databases such as DNA Databank of JAPAN (DDBJ), EMBL and GenBank,
  • Programs for homology search such as FASTA program and BLAST program.
  • database search services using the programs for searching the above databases are also available on Internet.
  • the nitrile hydratase to be used in the present invention can be found by using this type of service.
  • a polypeptide having at least 85%, preferably 90% or higher, more preferably 95% or higher identity to the amino acid sequences of SEQ ID NO: 2 ( ⁇ -subunit) or SEQ ID NO: 4 ( ⁇ -subunit) is a preferred polypeptide of the present invention constituting the nitrile hydratase to be used in the present invention.
  • percent identity means, for example, the value of percent identity in “Positive” using BLAST program. Specifically, “percent identity” of two amino acid sequences or of two nucleic acids is determined using the algorithm of Karlin and Altschul (Proc. Natl. Acad. Sci. USA 87: 2264-2268, 1990) modified as in Karlin and Altschul (Proc. Natl.
  • mutant ⁇ -subunit comprising the amino acid sequence of SEQ ID NO: 2 and mutant ⁇ -subunit comprising the amino acid sequence of SEQ ID NO: 4 both of which have mutations in their amino acid sequences
  • mutant polypeptides either or both of them may be mutant polypeptides.
  • the subunits of different origins can be combined with each other to form the protein complex of the present invention.
  • one of the two subunits is intended to be a mutant, it should be selected to be capable of constituting the protein complex, which has the above-mentioned physicochemical properties (1) and (2), with the other subunit.
  • mutants capable of constituting the protein complex that has the above-mentioned physicochemical properties (i) and (ii) are selected as well.
  • a mutant subunit is tested to assess whether it gives the required physicochemical properties in combination with the other subunit having the wild-type amino acid sequence when the result is positive, another mutant, which gives the required physicochemical properties, is selected for the partner subunit that is used in combination with the first mutant; thus the mutants are readily selectable.
  • the desirable mutants of the present invention have the above-mentioned physicochemical properties (a) and (b) or (1)-(7) as well as the above-mentioned physicochemical properties (i) and (ii).
  • Preferable enzymatically active material of the present invention includes a transformant of homologous or heterogonous host expressing the gene encoding nitrile hydratase, which has been prepared by genetic recombination techniques and treated products thereof.
  • the organism to be used for the transformation to express the nitrile hydratase gene of the present invention when the organism can be transformed with the recombinant vector containing polynucleotides encoding the respective subunits constituting the protein complex having the nitrile hydratase activity and can express the nitrile hydratase activity.
  • the polynucleotides encoding the respective subunits can be retaining in a single vector.
  • the polynucleotides for the respective subunits can also inserted separately in two types of vectors; the protein complex of the present invention can be expressed by the co-transformation of the vectors. Further, it is possible to obtain the protein complex of interest in vitro by combining the transformants each of which expresses a single subunit of the two.
  • Available microorganisms are those for which host-vector systems are available and include, for example:
  • bacteria such as the genus Escherichia , the genus Bacillus , the genus Pseudomonas , the genus Serratia , the genus Brevibacterium , the genus Corynebacteriun , the genus Streptococcus , and the genus Lactobacillus;
  • actinomycetes such as the genus Rhodococcus and the genus Streptomyces;
  • yeasts such as the genus Saccharomyces , the genus Kluyveromyces , the genus Schizosaccharomyces , the genus Zygosaccharomyces , the genus Yarrowia , the genus Trichosporon , the genus Rhodosporidium , the genus Pichia , and the genus Candida ; and,
  • fungi such as the genus Neurospora , the genus Aspergillus , the genus Cephalosporium , and the genus Trichoderma , etc.
  • Procedure for preparation of a transformant and construction of a recombinant vector suitable for a host can be carried out by employing techniques that are commonly used in the fields of molecular biology, bioengineering, and genetic engineering (for example, see Sambrook et al., “Molecular Cloning”, Cold Spring Harbor Laboratories).
  • nitrile hydratase of the present invention in a microorganism, it is necessary to introduce the polynucleotide into a plasmid vector or phage vector that is stable in the microorganism and to let the genetic information transcribed and translated.
  • a promoter a unit for regulating transcription and translation, is placed upstream of the 5′-end of the polynucleotide of the present invention, and preferably a terminator is placed downstream of the 3′-end of the polynucleotide.
  • the promoter and the terminator should be functional in the microorganism to be utilized as a host. Available vectors, promoters, and terminators for the above-mentioned various microorganisms are described in detail in “Fundamental Course in Microbiology (8): Genetic Engineering”, Kyoritsu Shuppan, specifically for yeasts, in “Adv. Biochem. Eng. 43, 75-102(1990)” and “Yeast 8, 423-488 (1992).”
  • available plasmids include pBR series and pUC series plasmids; available promoters include promoters derived from lac (derived from ⁇ -galactosidase gene), trp (derived from the tryptophan operon), tac and trc (which are chimeras of lac and trp), PL and PR of ⁇ phage, etc. Available terminators are derived from trpA, phages, rrnB ribosomal RNA, etc.
  • a vector pSE420D (described in Unexamined Published Japanese Patent Application No. (JP-A) 2000-189170), which is constructed by partially modifying the multicloning site of commercially available pSE420 (Invitrogen), can be preferably used.
  • available vectors are pUB110 series and pC194 series plasmids; the vectors can be integrated into host chromosome.
  • Available promoters and terminators are derived from apr (alkaline protease), npr (neutral protease), amy ( ⁇ -amylase), etc.
  • Pseudomonas For the genus Pseudomonas , there are host-vector systems developed for Pseudomonas putida and Pseudomonas cepacia .
  • plasmid vectors include pAJ43 (Gene 39, 281 (1985)). Promoters and terminators used for Escherichia coli can be utilized without any modification for Brevibacterium.
  • plasmid vectors such as pCS11 (JP-A Sho 57-183799) and pCB101 (Mol. Gen. Genet. 196, 175(1984) are available.
  • plasmid vectors such as pHV1301 (FEMS Microbiol. Lett. 26, 239 (1985)) and pGK1 (Appl. Environ. Microbiol. 50, 94 (1985)) can be used.
  • plasmid vectors such as pAM ⁇ 1 (J. Bacteriol. 137, 614 (1979)), which was developed for the genus Streptococcus , can be utilized; and promoters that are used for Escherichia coli are also usable.
  • Rhodococcus For the genus Rhodococcus , plasmid vectors isolated from Rhodococcus rhodochrous are available (J. Gen. Microbiol. 138, 1003 (1992)).
  • plasmids can be constructed in accordance with the method as described in “Genetic Manipulation of Streptomyces : A Laboratory Manual” (Cold Spring Harbor Laboratories (1985)) by Hopwood et al.
  • pIJ486 Mol. Gen. Genet. 203, 468-478, 1986
  • pKC1064 Gene 103,97-99 (1991)
  • pUWL-KS Gene 165, 149-150 (1995)
  • the same plasmids can also be utilized for Streptomyces virginiae (Actinomycetol. 11, 46-53 (1997)).
  • Saccharomyces for Saccharomyces cerevisiae , YRp series, YEp series, YCp series, and YIp series plasmids are available; integration vectors (refer EP 537456, etc.) which are integrated into chromosome via homologous recombination with multicopy-ribosomal genes, allow to introduce a gene of interest in multicopy and the gene incorporated is stably maintained in the microorganism; and thus, these types of vectors are highly useful.
  • integration vectors (refer EP 537456, etc.) which are integrated into chromosome via homologous recombination with multicopy-ribosomal genes, allow to introduce a gene of interest in multicopy and the gene incorporated is stably maintained in the microorganism; and thus, these types of vectors are highly useful.
  • Available promoters and terminators are derived from genes encoding ADH (alcohol dehydrogenase), GAPDH (glyceraldehyde-3-phosphate dehydrogenase), PHO (acid phosphatase), GAL ( ⁇ -galactosidase), PGK (phosphoglycerate kinase), ENO (enolase), etc.
  • ADH alcohol dehydrogenase
  • GAPDH glycoside dehydrogenase
  • PHO acid phosphatase
  • GAL ⁇ -galactosidase
  • PGK phosphoglycerate kinase
  • ENO enolase
  • plasmids are those such as 2- ⁇ m plasmids derived from Saccharomyces cerevisiae , pKD1 series plasmids (J. Bacteriol. 145, 382-390(1981)), plasmids derived from pGKl1 and involved in the killer activity, KARS ( Kluyveromyces autonomous replication sequence) plasmids, and plasmids (refer EP 537456, etc.) capable of being integrated into chromosome via homologous recombination with the ribosomal DNA. Promoters and terminators derived from ADH, PGK, and such are available.
  • plasmid vectors comprising ARS (autonomous replication sequence) derived from Schizosaccharomyces pombe and auxotrophy-complementing selectable markers derived from Saccharomyces cerevisiae (Mol. Cell. Biol. 6, 80 (1986)). Promoters such as ADH promoter derived from Schizosaccharomyces pombe are usable (EMBO J. 6, 729 (1987)) In particular, pAUR224 is commercially available from TaKaRa Shuzo.
  • plasmids originating from those such as pSB3 (Nucleic Acids Res. 13, 4267 (1985)) derived from Zygosaccharomyces rouxii are available; it is possible to use promoters such as PHO5 promoter derived from Saccharomyces cerevisiae and GAP-Zr (Glyceraldehyde-3-phosphate dehydrogenase) promoter (Agri. Biol. Chem. 54, 2521 (1990)) derived from Zygosaccharomyces rouxii.
  • promoters such as PHO5 promoter derived from Saccharomyces cerevisiae and GAP-Zr (Glyceraldehyde-3-phosphate dehydrogenase) promoter (Agri. Biol. Chem. 54, 2521 (1990)) derived from Zygosaccharomyces rouxii.
  • Pichia For the genus Pichia , host vector system where Pichia -derived genes involved in autonomous replication (PARS1 and PARS2) are used in Pichia pastoris and such has been developed (Mol. Cell. Biol. 5, 3376 (1985)), and thus high-density cultivation and strong promoters such as methanol-inducible AOX are usable (Nucleic Acids Res. 15, 3859 (1987)).
  • a Host-vector system has been developed for Pichia angusta (previously called Hansenula polyorpha ) among the genus Pichia .
  • Usable vectors include Pichia angusta -derived genes (HARS1 and HARS2) involved in autonomous replication, but they are relatively unstable.
  • Candida maltosa For the genus Candida , host-vector systems have been developed for Candida maltosa, Candida albicans, Candida tropicalis, Candida utilis , etc.
  • An autonomous replication sequence originating from Candida maltosa has been cloned (Agri. Biol. Chem. 51, 51,1587 (1987)), and a vector using the sequence has been developed for Candida maltosa .
  • a chromosome-integration vector with a highly efficient promoter unit has been developed for Candida utilis (JP-A Hei. 08-173170).
  • Aspergillus Aspergillus niger and Aspergillus oryzae have intensively been studied among fungi, and thus plasmid vectors and chromosome-integration vectors are available, as well as promoters derived from an extracellular protease gene and amylase gene (Trends in Biotechnology 7, 283-287 (1989)).
  • Trichoderma reesei For the genus Trichoderma , host-vector systems have been developed for Trichoderma reesei , and promoters such as that derived from an extracellular cellulase gene are available. (Biotechnology 7, 596-603(1989)).
  • the systems include those of insect such as silkworm (Nature 315, 592-594(1985)), and plants such as rapeseed, maize, potato and such are preferably usable.
  • insect such as silkworm (Nature 315, 592-594(1985))
  • plants such as rapeseed, maize, potato and such are preferably usable.
  • the culture of transformant and purification of nitrile hydratase from the transformant can be carried out by methods known to one skilled in the art.
  • Polynucleotides encoding nitrile hydratase to be used as inserts in the vector of the present invention include, for example, any one of the above polynucleotides shown in (A)-(E) and any one of the above polynucleotides shown in (a)-(e).
  • the polynucleotides encoding ⁇ -subunit and ⁇ -subunit of nitrile hydratase in the vector of the present invention are preferably linked in tandem.
  • the term. “preferably linked in tandem” means the linkage such that a common regulatory region directs the expression of these subunits. Such an arrangement is expected to enable more efficient expression of the subunits and more efficient formation of the protein complex having the enzymatic activity.
  • the polynucleotides for the respective subunits can also be inserted separately in two types of vectors; the protein complex can be expressed by the co-transformation of the two vectors.
  • the present invention also relates to transformants of the present invention retaining the vector in an expressible manner.
  • the vector of the present invention can be transformed into an arbitrary host, when the host can retain the vector in a functional form.
  • a host that is usable for this purpose includes, for example, E. coli.
  • the nitrile hydratase of the present invention, microorganism producing the nitrile hydratase, protein complex of the present invention, transformant producing the protein complex and treated products thereof are useful for the method to produce amides using nitrile compounds as the substrates.
  • the present invention provides a method for producing amides, the method comprising the step of recovering the amides by contacting nitrile compounds with the enzymatically active material selected from the group consisting of nitrile hydratase of the present invention, microorganism producing the nitrile hydratase, protein complex of the present invention, transformant producing the protein complex and processed products thereof.
  • the term “nitrile hydratase” means an enzyme having the above-mentioned physicochemical properties (a) and (b) or enzyme having the above-mentioned physicochemical properties (1)-(7).
  • the microorganism capable of producing the enzyme includes the strain Rhodococcus sp. Cr4 from which the enzyme is derived, microorganisms belonging to the genus Rhodococcus producing the enzyme of the present invention and transformed host microorganism containing the polynucleotide encoding the enzyme.
  • transformed host microorganism means a host microorganism capable of expressing the polynucleotides (A)-(E) encoding the above-mentioned ⁇ -subunit and/or polynucleotides (a)-(e) encoding ⁇ -subunit.
  • the above-mentioned host microorganism can produce the protein complex of the present invention consisting of ⁇ -subunit and ⁇ -subunit of the present invention.
  • the treated product of microorganism specifically includes microorganism of which cell membrane permeability has been modified by the treatment with a detergent or organic solvent such as toluene, cell-free extract obtained by lysing the cells by the treatment with glass beads or enzyme and material partially purified from the extract, etc.
  • the treated product of the enzyme includes the enzyme linked with insoluble carrier or with aqueous carrier molecule and the immobilized enzyme molecules prepared by immobilize entrapping, etc.
  • the enzymatically active material includes all materials having the enzymatic activity of nitrile hydratase of the present invention. Accordingly, as long as a material has the desired enzymatic activity, it is included in the enzymatically active material, regardless of its enzymatic purity and solubility.
  • the enzyme reaction constituting the amide-producing method of the present invention can be carried out by contacting the above-mentioned enzymatically active material with a reaction solution containing a nitrile compound as the substrate.
  • the enzymatically active material can be contacted with the substrate in an aqueous solvent, mixed solvent consisting of aqueous solvent and water-soluble organic solvent, or two-phase system with a water-insoluble solvent.
  • the aqueous solvent includes buffers having the buffering action at neutral pH such as phosphate buffer and Tris-HCl buffer.
  • the pH changes can be within a desirable range during the reaction by using an acid and alkali, no buffer is needed.
  • the organic solvent immiscible with water includes, for example, ethyl acetate, butyl acetate, toluene, chloroform, n-hexane and isooctane, etc.
  • the reaction can be conducted in a mixed solvent, which consists of an aqueous solvent, and organic solvent such as ethanol, acetone, dimethylsulfoxide and acetonitrile.
  • the enzymatically active material is present in the aqueous phase in which the material is used without any other solvent or combined with water or a buffer.
  • the substrate compound can be dissolved in an aqueous solvent such as water, buffer and ethanol, and supplied to the reaction system.
  • the substrate constitutes a single-phase reaction system.
  • the reaction of the present invention can be carried out by using immobilized enzyme, membrane reactor, etc.
  • the transformant of the present invention can be used in the form of the culture, cells separated from the culture medium by filtration, centrifugation or the like, or cells resuspended in buffer, water, or the like after they are separated by centrifugation and washed.
  • the separated cells can be used in a state as they are recovered, as their disrupts, as treated with acetone or toluene, or as lyophilizate.
  • the culture medium of the transformant can also be used after it is separated from the transformant by the usual methods.
  • Forms of contacting the enzymatically active material with the reaction solution are not limited to these Examples.
  • the reaction solution means a solution consisting of the substrate dissolved in an appropriate solvent that provides suitable environment for the expression of enzymatic activity.
  • nitrile compound there is no limitation on the type of nitrile compound to be used in the method for producing amides using the nitrile hydratase of the present invention.
  • the following nitrile compounds can be used in the method of the present invention.
  • Aromatic nitrites Aromatic nitrites
  • particularly preferable nitrile compounds include ⁇ -hydroxynitrile compound.
  • ⁇ -hydroxynitrile compound there is no limitation on the type of ⁇ -hydroxynitrile compound. More specifically, for example, the compound represented by the above formula (1) can be used.
  • R represents, substituted or unsubstituted alkyl group, substituted or unsubstituted alkenyl group, substituted or unsubstituted cycloalkyl group, substituted or unsubstituted alkoxy group, substituted or unsubstituted aryl group, substituted or unsubstituted aryloxy group, substituted or unsubstituted saturated or unsaturated heterocyclic group.
  • ⁇ -hydroxyamide can be produced from the ⁇ -hydroxynitrile compounds.
  • the heterocyclic group includes the groups having at least one of nitrogen, oxygen and sulfur as the heteroatom.
  • the substituent includes, for example, alkyl group, alkoxy group acyl group, aryl group, aryloxy group, halogens such as chloride and bromide, hydroxy group, amino group, nitro group, thiol group, etc.
  • the substituted product means compounds having the substituents as exemplified above.
  • the substrate compound having aromatic ring and heterocycle include the following exemplary compounds and substituted products thereof.
  • nitrile compounds represented by the ⁇ -hydroxynitrile compound of formula (1) are decomposed into aldehyde and hydrocyanic acid in a polar solvent.
  • the ⁇ -hydroxynitrile compound of formula (1) is converted to the aldehyde and hydrocyanic acid of the following formula (2).
  • cyanide and aldehyde derived from hydrocyanic acid may give some damage to the enzyme polypeptide. Accordingly, previously known nitrile hydratases cannot hydrate sufficient amounts of ⁇ -hydroxynitrile compound due to their decreased enzymatic activity, and thus do not provide enough yields of the products. However, the nitrile hydratase of the present invention retains the enzymatic activity even in the presence of cyanide or aldehyde.
  • the enzyme can utilize nitrile compounds generated from aldehydes and hydrocyanic acids as the substrate. Accordingly, by the inventive method for producing ⁇ -hydroxyamide, the compound of formula (1) can be supplied from the aldehyde compound and hydrocyanic acid represented by the following formula (2).
  • the amide-producing method of the present invention is preferably conducted in the presence of a divalent metal ion.
  • the divalent metal ion contributes to the activity of nitrile hydratase of the present invention.
  • the preferable divalent metal ions include nickel ion and cobalt ion. These ions can be added to the reaction solution as an appropriate aqueous salt. Specifically, the ion can be added as a chloride salt.
  • the hydration or hydrolysis of nitrile compound can be achieved by contacting the enzymatically active material of the present invention with a substrate compound, or a mixture of aldehyde and hydrocyanic acid represented by formula (2), which can be converted to a substrate compound in an aqueous solvent such as water or buffer.
  • the reaction solution preferably contains divalent metal ions.
  • hydrolysis means the reaction where, from a compound in which a substituent is linked with the nitrile group, the substituent is cleaved off by hydrolysis. Both reactions are included by the amide-producing method of the present invention.
  • the concentration of substrate compound in the reaction solution can correspond to, for example, 0.1-10 w/w % in general, preferably 0.2-5.0 w/w %, in the case of ⁇ -hydroxynitrile.
  • the substrate can be added once at the start of reaction, but it is preferable to add the substrate continuously or discontinuously to prevent the substrate concentration from being too high.
  • a detergent can be added to the reaction solution.
  • 0.1-5.0 w/w % Triton X-100 or Tween 60 can be use as the detergent.
  • a mixed solvent containing an organic solvent can be used effectively.
  • the reaction efficiency can be improved by adding methanol, ethanol, dimethylsulfoxide, and such.
  • the reaction of the present invention can be achieved in an organic solvent insoluble with water or two-phase system consisting of aqueous solvent and organic solvent insoluble with water.
  • the organic solvent immiscible with water that is usable includes, for example, ethyl acetate, butyl acetate, toluene, chloroform, n-hexane, cyclohexane, octane or 1-octanol, etc.
  • the efficient enzymatic reaction can be achieved by using nitrile hydratase of the present invention at an enzyme concentration, for example, of 1 mU/mL-100 U/mL, preferably 100 MU/mL or higher.
  • an enzyme concentration for example, of 1 mU/mL-100 U/mL, preferably 100 MU/mL or higher.
  • the amount of microorganism to be used relative to that of the substrate preferably ranges from 0.01 to 5.0 w/w % as dry cells.
  • the enzymatically active material such as an enzyme and cells can be contacted with the substrate by dissolving or dispersing them in a reaction solution.
  • the enzymatically active material immobilized by the techniques of chemical linking or entrapment. Further, the reaction can be carried out in a state where the substrate solution is isolated from the enzymatically active material, with a porous membrane, through which the substrate is permeable, but which limits the permeation of the enzyme molecule or the cells.
  • the reaction can be carried out typically at a temperature ranging from the freezing point to 50° C., preferably at 10-30° C., for 0.1-100 hours.
  • There is no limitation on the pH of reaction solution when the enzymatic activity can be maintained. Since the optimal pH of nitrile hydratase of the present invention ranges from 5.5 to 6.5, it is preferable to adjust the pH of reaction solution within this range.
  • the nitrile compound is converted to a corresponding amide by the hydration or hydrolysis action of the microorganism, and accumulates in the reaction solution.
  • the produced amide can be recovered and purified from the reaction solution by appropriate methods. Specifically, for example, the amide can be recovered and purified by the combined use of typical methods such as ultrafiltration, concentration, column chromatography, extraction, treatment with activated charcoal, distillation, etc.
  • the present invention provides a method for stabilizing the activity of nitrile hydratase in the presence of the nitrile compound, where the method is characterized by coexistence of divalent metal ions. Further, the present invention provides a method for producing amide, the method comprising the step of reacting the nitrile hydratase to the nitrile compound in the presence of divalent metal ion and recovering the amide generated.
  • the present invention is based on the finding that the presence of divalent metal ion in the reaction system markedly suppresses the decrease in the enzymatic activity of nitrile hydratase activity.
  • nitrile hydratase there is no limitation on the origin of nitrile hydratase. Namely, any enzyme can be used, when the enzyme has the activity of acting on and hydrating the nitrile group of nitrile compound, and converting the nitrile group to the amide group.
  • the nitrile hydratase which hydrates ⁇ -hydroxynitrile to the corresponding amide, is the preferred enzyme in the present invention.
  • the divalent metal ion of the present invention achieves the effect of suppressing the decrease in the nitrile hydratase activity, for example, by the following mechanism:
  • the binding of metal ion to the cyanohydrin structure of substrate compound may enhance the enzyme reaction.
  • the binding of metal ion may prevent the dissociation of cyanohydrin.
  • the concentration of cyanide ion is reduced in the reaction solution.
  • the reduced cyanide ion concentration lowers the inhibitory effect on the enzyme, the enzymatic activity is raised.
  • Known enzymes capable of hydrating nitrile compounds to the corresponding amides include, for example, the enzymes derived form the following microorganism (see JP-A Hei 04-040899).
  • Rhodococcus The genus Rhodococcus
  • the genus Brevibacterium The genus Brevibacterium
  • the genus Nocardia The genus Nocardia.
  • microorganisms include the following:
  • Arthrobacter sp. HR1 (FERM BP-3323)
  • Rhodococcus sp. HT40-6 (FERM P-11774)
  • nitrile hydratase of the present invention that can be obtained from the above-mentioned Rhodococcus sp. Cr4 (FERM BP-6596) is also one of the preferred enzyme of the present invention.
  • the nitrile compound to be used in the amide-producing method of the present invention there is no limitation on the nitrile compound to be used in the amide-producing method of the present invention. More specifically, the preferred substrates include, for example, compounds illustrated in the above description for the method for producing ⁇ -hydroxyamide with the nitrile hydratase of the present invention.
  • the enzyme capable of stereospecifically hydrating or hydrolyzing nitrites or a microorganism containing the enzyme is used in the reaction.
  • the stereospecific ⁇ -hydroxyamide or ⁇ -hydroxy acid can be obtained much more advantageously than by the previous methods producing it through the step of optical resolution or racemization.
  • the hydration or hydrolysis of nitrile compound is carried out, for example, by contacting the nitrile hydratase with the ⁇ -hydroxynitrile represented by formula (1) in an aqueous solvent such as water or buffer.
  • an aqueous solvent such as water or buffer.
  • the reaction can also be carried out in the presence of the aldehyde and hydrocyanic acid represented by formula (2), which can be converted to ⁇ -hydroxynitrile.
  • the nitrile hydratase may be a lysate of microorganism capable of producing the enzyme, crude enzyme, or products obtained by immobilizing the materials, in addition to the purified enzyme.
  • the divalent metal ion may be added in the reaction solution at a concentration of 0.1 mM-1 M, generally 0.5-100 mM, preferably 1-10 mM.
  • metal ion there is no limitation on the type of metal ion to be used in the present invention, when the ion can be effective for maintaining the activity of nitrile hydratase.
  • cobalt ion and nickel ion are preferred metal ions, which can keep the activity of nitrile hydratase high.
  • These metal ions can be added as salts such as chloride salts in the reaction solution.
  • the reaction conditions can be adjusted appropriately depending on the properties of substrate compounds to be used in combination with the nitrile hydratase, in addition to the presence of divalent metal ion.
  • the type of substrate compound and reaction conditions to be used in the reaction can be adjusted based on the exemplary conditions illustrated for the method for producing the ⁇ -hydroxyamide using the above-mentioned nitrile hydratase of the present invention.
  • % for concentration denotes weight per volume percent unless otherwise specified.
  • FIG. 1 shows the determination of molecular weight of nitrile hydratase of the present invention by SDS-PAGE.
  • the abscissa indicates the relative mobility and the ordinate indicates the molecular weight (kDa).
  • FIG. 2 shows an elution profile of nitrile hydratase of the present invention by column chromatography with butyl-Toyopearl 650M.
  • White square ( ⁇ ) represents OD 280
  • closed circle ( ⁇ ) represents the activity
  • hyphen (-) represents (NH 4 ) 2 SO 4 .
  • FIG. 3 shows the determination of molecular weight of nitrile hydratase of the present invention by gel filtration.
  • the abscissa indicates retention time (minute) and the ordinate indicates the molecular weight (kDa).
  • FIG. 4 shows the Km value of nitrile hydratase of the present invention for HMBN, which was determined with an S-V plot.
  • FIG. 5 the Km value of nitrile hydratase of the present invention for HMBN, which was determined with a Lineweaver-Burk plot.
  • FIG. 6 shows the Km value of nitrile hydratase of the present invention for 3-cyanopyridine, which was determined with an S-V plot.
  • FIG. 7 shows the Km value of nitrile hydratase of the present invention for 3-cyanopyridine, which was determined with a Lineweaver-Burk plot.
  • FIG. 8 shows the effect of temperature on the activity of nitrile hydratase of the present invention.
  • FIG. 9 shows the thermal stability of nitrite hydratase of the present invention.
  • FIG. 10 shows the effect of pH on the activity of nitrile hydratase of the present invention.
  • FIG. 11 shows the pH stability of the nitrile hydratase of the present invention.
  • FIG. 12 shows the organization of gene cluster in Rhodococcus sp. Cr4.
  • the nucleotide sequences indicated by capital letters correspond to the ORFs.
  • the names of subunits encoded the ORFs are shown at the right of the nucleotide sequences.
  • FIG. 13 shows the amino acid comparison between ⁇ -subunits of nitrile hydratase from Rhodococcus sp. Cr4 and Rhodococcus rhodochrous J1.
  • Rhodococcus sp. Cr4 top panel; CrNH- ⁇
  • Rhodococcus rhodochrous J1 bottom panel; J1 L-a
  • identical amino acids are represented by an asterisk and distinct amino acids are represented by a space.
  • FIG. 14 shows the amino acid comparison between ⁇ -subunits of nitrile hydratase from Rhodococcus sp. Cr4 and Rhodococcus rhodochrous J1.
  • Rhodococcus sp. Cr4 top panel; CrNH- ⁇
  • Rhodococcus rhodochrous J1 bottom panel; J1 L- ⁇
  • identical amino acids are represented by an asterisk and distinct amino acids are represented by a space.
  • the standard assay method for the nitrile hydratase activity used in the following Examples as follows.
  • the standard composition of reaction solution for the enzyme reaction is shown in Tables 1 and 2.
  • the enzyme reaction is initiated by adding 3-cyanopyridine or 2-hydroxy-4-methylthiobutyronitrile (HMBN) as the substrate compound.
  • 3-cyanopyridine incubation was continued at 20° C. for 10 minute, in the case of HMBN, at 20° C. for 15 minutes.
  • the generated nicotinamide or HMBAm was quantified by HPLC, and the nitrile hydratase activity was calculated.
  • the conditions of HPLC assay HMBAm were the same as that for HMBN.
  • 1 U was defined as the enzyme quantity capable of producing 1 ⁇ mol nicotinamide with the standard composition of reaction solution at 20° C. for 1 minute; or 1 U was defined as the enzyme quantity capable of producing 1 ⁇ mol HMBAm with the standard composition of reaction solution at 20° C. for 1 minutes.
  • the quantity of protein was determined according to Bradford method (Bradford, M., Anal, Biochem., 72, 248(1976)) with a protein assay kit from Bio-Rad.
  • DEAE-Sephacel and Butyl-Toyopearl 650 M used was provided by Pharmacia; bovine serum albumin, from Bio-Rad; molecular weight marker for SDS-PAGE, from Pharmacia; molecular weight marker for HPLC, from Oriental yeast. Unless otherwise specified, other reagents used were commercially available special-grade reagents.
  • the pre-culture medium of the following composition was aliquoted in 5 ml into each test tube (25 ⁇ 200 mm); a silicone plug was placed in the tube, followed by sterilization by autoclaving. After the tube was cooled, a bacterial strain was inoculated with a platinum loop, and then cultured with shaking at 28° C. for two days.
  • Pre-culture medium (pH 7.0): Polypeptone 5.0 g Meat extract 5.0 g NaCl 2.0 g Yeast extract 0.5 g Distilled water 1.0 L
  • the pre-culture was transferred into 20-ml main-culture medium autoclaved in a 500-ml Sakaguchi flask.
  • a feeding needle After 24-hour culture, and then the incubation was continued with shaking at 33° C. for two days.
  • Main-culture medium (pH 7.0): Acetamide 7.5 g Glucose 10.0 g C.S.L. 10.0 g Yeast extract 1.0 g MgSO 4 7H 2 O 0.5 g K 2 HPO 4 1.0 g CoCl 2 6H 2 O 20.0 mg Distilled water 1.0 L 3.
  • the bacterial cells corresponding to 500 ml of the culture liquid were suspended at a 5-times higher cell density in 50 mM phosphate buffer/44 mM n-butyric acid (pH 7.0).
  • the suspended bacterial cells were homogenized with a sonicator 201 M (Kubota) with 150 W at 4° C. or a lower temperature for 60 minutes.
  • the culture was separated into the supernatant and precipitating fractions by centrifuge at 13,000 rpm for 20 minutes. The supernatant was used as cell-free extract for the following purification.
  • Ammonium sulfate was added to the cell-free extract at 30% saturation. The mixture was neutralized to pH 7.0 with 10%(v/v) ammonium water, and then stirred at 4° C. for three hours. The solution was then fractionated into the supernatant and precipitating fractions by centrifuging at 13,000 rpm for 30 minutes. Ammonium sulfate was added to the supernatant fraction at 60% saturation. The mixture was neutralized to pH 7.0, and then stirred at 4° C. for three hours. The solution was then fractionated into the supernatant and precipitating fractions by centrifuging. Further, ammonium sulfate was added to the supernatant fraction at 80% saturation.
  • the enzyme solution which had been sufficiently dialyzed against the same buffer, loaded onto a column ( ⁇ 13 ⁇ 216 mm) of DEAE-Sephacel sufficiently equilibrated with 10 mM phosphate buffer/44 mM n-butyric acid (pH 7.0). The column was then washed with the same buffer. Then, elution was carried out with the following elution buffer, and the enzymatic activity in the fractions was assessed. The nitrile hydratase activity was found in the fractions of No.74-80 when eluted with 10 mM phosphate buffer/44 mM n-butyric acid (pH 7.0)/0.3 M KCl. The volume of each elution buffer used was approximately 3 times as much as-the volume of carrier.
  • the purified enzyme obtained was analyzed by SDS-PAGE.
  • SDS-PAGE was carried out according to Laemmli's method (Laemmli, U.K.: Nature, 227, pp680, 1970). Namely, electrophoresis was performed in a 12% polyacrylamide slab gel with Tris/glycine buffer. An equal-volume mixture of enzyme solution and sample buffer was heat-treated at 90° C. for about 10 minutes. The gel was stained with Coomassie brilliant blue R-250; destaining was carried out with ethanol/acetic acid/dH 2 O (2/3/6, by vol.). The result was shown in FIG. 1 .
  • the purified enzyme was revealed to consist of ⁇ -subunit (26.8 kDa) and ⁇ -subunit (29.5 kDa).
  • the nitrile hydratase was purified according to the method as described above. First, the suspension of homogenized bacterial cells was fractionated into the supernatant and precipitating fractions by centrifugation. The activity was contained in the supernatant fraction, and thus the supernatant was used as a cell-free extract. The cell-free extract was fractionated with ammonium sulfate, and, as a result, the activity was contained in the fractions of which ammonium sulfate concentration is 30-60% saturation. The active fractions were subjected to column chromatography with DEAE-Sephacel. The active fractions (No.
  • the conditions used for the HPLC analysis of nitrile hydratase for the molecular weight is shown in Table 4.
  • the molecular weight marker used was from Oriental Yeast.
  • the Km value for HMBN was determined from the S-V plot ( FIG. 4 ) and Lineweaver-Burk plot ( FIG. 5 ). 0.0585 U of the enzyme was added to solutions containing HMBN at various concentrations and the reactions were incubated at 20° C. for 15 minutes. The reactions were analyzed by HPLC. The Km value for HMBN was determined to be 1.43 mM from the Lineweaver-Burk plot, and this indicates that the enzyme has high affinity for HMBN.
  • the Km value for 3-cyanopyridine was determined from the S-V plot ( FIG. 6 ) and Lineweaver-Burk plot ( FIG. 7 ).
  • 0.0265 U of the enzyme was added to solutions containing 3-cyanopyridine at various concentrations and the reactions were incubated at 20° C. for 15 minutes. The reactions were analyzed by HPLC.
  • the Km value for 3-cyanopyridine was determined to be 1.38 mM from the Lineweaver-Burk plot, and this indicates that the enzyme also has high affinity for 3-cyanopyridine.
  • the optimal temperature and thermal stability of the purified enzyme was evaluated. With the standard composition of reaction solution as shown in Table 1, 2.92 U enzyme was incubated at each temperature (10, 20, 30, 35, 40, 45, 50, 55, 60, or 65° C.) for 15 minutes to assess the optimal temperature ( FIG. 8 ). The temperature, at which the reaction rate with the enzyme was maximized, was 45° C. Further, 2.92 U of the enzyme was heat-treated at each temperature (10, 20, 30, 40, 50, or 60° C.) for 30 minutes, and then the thermal stability was assessed under the standard reaction conditions. After the enzyme treated at 50° C., the remaining activity was 77% ( FIG. 9 ).
  • the optimal pH and pH stability of the purified enzyme was evaluated. With the standard composition of reaction solution as shown in Table 1, 2.92 U enzyme was incubated in each buffer (buffer of sodium citrate or Tris-HCl at a final concentration of 50 mM), instead of phosphate buffer, at 20° C. for 15 minutes to determine the optimal pH. The optimal pH was 6.0 ( FIG. 10 ).
  • the substrate specificity of the enzyme was examined. With the standard composition reaction solution shown in table 2, 2.92 U enzyme was incubated with various types of substrates, instead of 3-cyanopyridine; the generation of amides in the reaction solution was analyzed by HPLC. Thus, the relative activity was determined. The types of substrate compounds added, final concentrations thereof, reaction times, and HPLC conditions used for the analyses of products corresponding to the respective substrates are shown in Table 6. The HPLC conditions (1)-(9) are indicated next to Table 6.
  • the purified nitrile hydratase showed substrate specificity that was very similar to that of resting cell reaction; malononitrile, n-butyronitrile and 2-cyanopyridine were suitable substrates.
  • the relative activity for HMBN was 154%, when the activity for 3-cyanopyridine was taken as 100% (Table 5).
  • Inhibitors to the enzyme were studied. With the standard composition of reaction solution shown in Table 1, various inhibitors were added at a final concentration 1.0 mM or 0.1 mM to the solutions containing 2.92 U enzyme; after the mixtures were incubated at 20° C. for 10 minutes, the reaction was carried out for 15 minutes. The enzyme activity was markedly inhibited by carbonyl reagents such as phenyl hydrazine and hydroxylamine (Table 7).
  • Ni ++ or Co ++ ion was added at a concentration of 0-8.0 mM to a solution containing 2.92 U enzyme; its effect on the enzymatic activity was studied.
  • Ni ++ or Co ++ ion was added at a concentration of 1.0-2.0 mM, the enzymatic activity was enhanced twice or 1.5 times.
  • the metal ions were added at higher concentration than above-mentioned one, the enhancement was impaired (Tables 9 and 10).
  • the enzyme incubated with nickel ion at 20° C. for five minutes, and the enzymatic activity was assayed.
  • the enzymatic activity of nitrile hydratase of the present invention was enhanced twice by adding Ni ++ ion to the reaction system at a concentration of 1.0 mM. Then, with the standard composition of reaction solution shown in table 2, various types of substrates, instead of 3-cyanopyridine, were added to the solutions containing 2.92 U enzyme to study their effects on the activity. The activity was enhanced twice only when HMBN or mandelonitrile was used as the substrate. The addition of Ni ++ ion was assumed to specifically enhance the reaction efficiency when the substrate was a ⁇ -hydroxynitrile (Table 11).
  • N-terminal amino acid sequences of ⁇ -subunit and ⁇ -subunit of nitrile hydratase from Rhodococcus sp. Cr4 strain were analyzed in a Procise Sequencer Model 470A from Applied Biosystem.
  • the sequence of N-terminal 15 residues of ⁇ -subunit was TAHNPVQGTFPRSNE (SEQ ID NO: 5); that of ⁇ -subunit was MDGIHDLGGRAGLGP (SEQ ID NO: 6).
  • the preculture medium of the following composition was aliquoted in 5 ml into each test tube (25 ⁇ 200 mm); a silicone plug was placed in the tube, followed by sterilization by autoclaving.
  • Pre-culture medium (pH 7.0): Polypeptone 5.0 g Meat extract 5.0 g NaCl 2.0 g Yeast extract 0.5 g Distilled water 1.0 L
  • Rhodococcus sp. Cr4 or Rhodococcus rhodochrous ATCC 332878 strain was inoculated with a platinum loop into the pre-culture medium, and then cultured with shaking at 28° C. for two days. Then, the pre-culture of Rhodococcus sp. Cr4 was transferred into main-culture medium 1; that of Rhodococcus rhodochrous ATCC 332878 was transferred into main-culture medium 2. Both were cultured with shaking at 33° C. for two days. The bacterial cells were harvested and washed, and then the reaction shown in Table 14 was carried out to study the effect of NiCl 2 and CoCl 2 added.
  • NiCl 2 or CoCl 2 was added to the reaction system at a final concentration of 1 mM, as seen Table 15, the coexisting NiCl 2 or CoCl 2 enhanced the nitrile hydratase activity, and thus the addition of the ions had positive effect.
  • Main-Culture Medium 1 (for Rhodococcus sp. Cr4) (pH 7.0):
  • ⁇ -caprolactam 5.0 g Glucose 10.0 g C.S.L. 10.0 g Yeast extract 1.0 g MgSO 4 7H 2 O 0.5 g K 2 HPO 4 1.0 g CoCl 2 6H 2 O 20.0 mg Distilled water 1.0 L
  • Each medium was aliquoted in 20 ml into 500-ml Sakaguchi flasks, and then used after sterilized by autoclaving.
  • Rhodococcus sp. Cr4 nitrile hydratase the quantity of enzyme used was 288 U/ml; in the case of Rhodococcus rhodochrous J1 nitrile hydratase, the quantity of enzyme used was 61 U/ml.
  • the nitrile hydratase of Rhodococcus rhodochrous J1 was completely inhibited in the presence of 1 mM cyanide ion, but the nitrile hydratase of Rhodococcus sp. Cr4 retained 47% of the activity in the presence of 1 mM cyanide ion and 17% of the activity in the presence of 5 mM cyanide ion.
  • the nitrile hydratase purified from Rhodococcus sp. Cr4 was subjected to SDS-PAGE.
  • the protein was electro-transferred onto a PVDF membrane with a Horiz-Blot Electrophoresis Apparatus Model AE6678-P (ATTO), and then stained with amide black. Portions corresponding to ⁇ -subunit and ⁇ -subunit were cut off, and each of the subunit proteins were subjected to automatic Edman degradation in a gas-phase peptide sequencer model 473A (Applied Biosystem), and thus PTH amino acid derivatives were obtained.
  • the N-terminal amino acid sequences of ⁇ -subunit and ⁇ -subunit were analyzed in a PTH amino acid derivative analyzer model 120A (Applied Biosystem).
  • the N-terminal sequence was TAHNPVQGTFPRSNE (SEQ ID NO: 5) for ⁇ -subunit; MDGIHDLGGRAGLGPI (SEQ ID NO: 7) for ⁇ -subunit.
  • the sequence of ⁇ -subunit exhibited 93% identity (L at amino acid residue 10 in J1) to the N-terminal sequence of ⁇ -subunit of low-molecular-weight nitrile hydratase from Rhodococcus rhodochrous J1; the sequence of ⁇ -subunit did 100% identity to that of the equivalent from Rhodococcus rhodochrous J1.
  • N-terminal amino acid sequences of nitrile hydratase ⁇ -subunit and ⁇ -subunit from Rhodococcus rhodochrous J1 are nearly identical to those from Rhodococcus sp. Cr4. Further, it is known that ⁇ -subunits of previously reported nitrile hydratases share the highly homologous primary structures. Thus, cloning of the nitrile hydratase gene of Rhodococcus sp. Cr4 was carried out using, as a probe DNA, a gene fragment from ⁇ -subunit of nitrile hydratase of Rhodococcus rhodochrous J1.
  • a gene fragment form ⁇ -subunit of nitrile hydratase of Rhodococcus rhodochrous J1 was used to evaluate whether the entire region of nitrile hydratase gene of Rhodococcus sp. Cr4 was successfully cloned.
  • Rhodococcus rhodochrous J1 There two types of nitrile hydratases in Rhodococcus rhodochrous J1, namely, the low molecular weight form and high molecular weight form. Therefore, firstly, regions of the primary structure of low molecular weight nitrile hydratase gene, of which sequences are different from those corresponding regions of the high molecular weight form, in Rhodococcus rhodochrous J1, were selected. Specifically, the respective amino acid sequences selected are described below.
  • PCR primers to be used for specifically amplifying a part of the low molecular weight nitrile hydratase gene, were actually designed. Finally, the primers comprising the following nucleotide sequences were designed.
  • the genomic DNA was prepared from Rhodococcus rhodochrous J1 according to the method of Ausbel et al. (Ausbel, F M et al: UNIT 2.4, Preparation of Genomic DNA from Bacteria in Current Protocols in Molecular Biology (John Wiley and Sons, New York) 1987). Further, the genomic DNA was also prepared from culture cells of Rhodococcus sp. Cr4 by the same method.
  • a part of the low molecular weight nitrile hydratase gene of Rhodococcus rhodochrous J1 was amplified by PCR using Taq DNA 30 (Takara Shuzo) and cycles of: denaturation at 94° C. for 1 minutes, annealing at 60° C. for 90 seconds, and extension at 72° C. for 90 seconds using 10 ng of the genomic DNA obtained as a template. Amplified DNA fragment was subjected to agarose gel electrophoresis and then collected from the gel by using a RECOCHIP (Takara Shuzo).
  • PCR amplification using the primers for the amplification of ⁇ -subunit gene fragment and the genomic DNAs from Rhodococcus rhodochrous J1 and Rhodococcus sp. Cr4 as templates gave amplification products of about 450 bp from both strains.
  • the genomic DNA from Rhodococcus sp. Cr4 was used as the template, the efficiency of PCR amplification was lower.
  • the amplified fragment by using the genomic DNA of Rhodococcus rhodochrous J1 as the template was used as the probe DNA for the subsequent experiments.
  • the amplified DNA fragment derived from Rhodococcus rhodochrous J1 was labeled with digoxigenin according to the random priming method with a DIG-labeling kit (Boehringer Manheim).
  • the digoxigenin-labeled DNA fragment was used as the probe DNA in the subsequent Southern blot analysis and colony hybridization.
  • the genomic DNA of Rhodococcus sp. Cr4 was digested with various restriction enzymes, and analyzed by Southern blotting using the probe DNA from ⁇ -subunit of nitrile hydratase derived from Rhodococcus rhodochrous J1. 10 ⁇ g of the genomic DNA from Rhodococcus sp. Cr4 was completely digested with PstI, SphI and BanIII (Toyobo), and then 1- ⁇ g aliquots of the digests were electrophoresed on a 0.8% agarose gel. The DNA was transferred onto a nylon membrane NY13N (Schleicher & Schuel) in a vacuum blotter model 785 (Bio-Rad) according to the attached manual.
  • the DNA was immobilized on the membrane in an autocrosslinker CL-1000 (BM Equipment)
  • CL-1000 BM Equipment
  • the resulting membrane was hybridized with the probe DNA at 60° C. overnight, and then washed at room temperature for 5 minutes and then at 60° C. for 15 minutes.
  • the chemical luminescent light by CDP-Star was exposed onto a Fuji RX film for signal detection by using a DIG detection kit (Boehringer Manheim) according to the attached manual.
  • the analysis of Southern blotting revealed single bands of 3.2 kb, 4.2 kb and 7.8 kb in the lanes of the genomic DNA from Rhodococcus sp. Cr4 digested with PstI, SphI and BanIII, respectively.
  • the nitrile hydratase gene of Rhodococcus sp. Cr4 was found to be a single-copy gene.
  • the nitrile hydratase gene of Rhodococcus sp. Cr4 was cloned from a genomic library prepared from the PstI digest.
  • a plasmid pBluescript II SK (+) (Stratagene) was digested with PstI, and then dephosphorylated by bacterial alkaline phosphatase (Takara Shuzo).
  • the genomic DNA of Rhodococcus sp. Cr4 was digested with PstI, and then ligated to the above pBluescript II SK (+) by using T4 DNA ligase (Takara Shuzo). Then, E. coli DH5 ⁇ was transformed by above-mentioned vector.
  • Colony hybridization was performed according to the method of Sambrook et al. (Molecular cloning (Cold Spring Harbor Laboratory Press, Cold Spring Harbor) 1989). Specifically, a nitrocellulose filter was placed on an LB agar plate containing 50 ⁇ g/ml ampicillin, and the transformants obtained were spread thereon. The plate was incubated at 37° C. overnight. After the colonies were formed, a replica filter prepared was placed on the agar plate and cultured thereon. After the colonies were formed on the replica filter., the filter was transferred onto an agar plate containing 200 ⁇ g/ml chloramphenicol. The plate was incubated at 37° C. for 24 hours. The filter was treated with SDS and alkali to lyse the colonies. Then, colony hybridization was carried out under the same conditions as used in the analysis of Southern blotting. Thus, a positive clone was obtained from about 2000 colonies.
  • the plasmid was prepared by the alkali-SDS method; the plasmid was named pNHCr4P.
  • the plasmid PNHCr4P contained an insert of about 3.2-kb DNA fragment.
  • a probe DNA was prepared by PCR using the primers for the amplification of the ⁇ -subunit gene fragment and the genomic DNA of Rhodococcus rhodochrous J1 as a template. With this probe DNA, pNHCr4P was analyzed by Southern blotting. As a result, the insert fragment of about 3.2 kb containing the ⁇ -subunit gene was confirmed.
  • the nucleotide sequence of insert DNA fragment (about 3.2 kb) in pNHCr4P was analyzed by the dideoxy chain termination method using a DNA sequencer model 377A (Applied Biosystem) and Taq primer cycle sequencing kit (Applied Biosystem). The determined nucleotide sequence is shown in SEQ ID NO: 16. The sequencing result showed that the size of PstI insert DNA fragment in pNHCr4P was 3205 bp and that an ORF was presence ( FIG. 12 ). The organization of gene cluster in Rhodococcus sp.
  • the present invention provides a nitrile hydratase capable of producing 2-hydroxy-4-methylthiobutyroamide from 2-hydroxy-4-methylthiobutyronitrile as a substrate.
  • 2-hydroxy-4-methylthiobutyroamide is a useful compound as a feed additive (a methionine substitute).
  • the present invention provides the nitrile hydratase capable of enzymatically producing 2-hydroxy-4-methylthiobutyroamide, but also the encoding gene was cloned in the present invention.
  • the nitrile hydratase of the present invention can be expressed at high levels in appropriate host cells transformed with the gene encoding the nitrile hydratase provided by the present invention.
  • the transformants themselves that can be obtained according to the present invention, or the enzyme protein obtained from the transformants are useful for enzymatically producing 2-hydroxy-4-methylthiobutyroamide.
  • the nitrile hydratase of the present invention is excellent retaining the high activity even when it is a genetic recombinant.
  • the nitrile hydratase of the present invention retains the high enzymatic activity in the presence of cyanide or aldehyde.
  • cyanide or aldehyde In a polar solvent, ⁇ -hydroxynitrile as the substrate compound is decomposed to hydrocyanic acid and aldehyde. Hydrocyanic acid is converted to cyanide, which often reduces the enzymatic activity. Aldehyde also gives damages to the protein and reduces the enzymatic activity.
  • cyanide and aldehyde reduces the enzymatic activity.
  • the hydratase of the present invention retains the enzymatic activity even in the presence of cyanide or aldehyde. Therefore, ⁇ -hydroxynitrile produced from aldehyde and hydrocyanic acid can be used as the substrate.
  • the nitrile hydratase of the present invention is useful for the method for producing amides using ⁇ -hydroxynitrile as starting material.

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